Methods for in vitro joining and combinatorial assembly of nucleic acid molecules
Inventors
Gibson, Daniel G. • Smith, Hamilton O. • HUTCHISON, Clyde A. • Young, Lei • Venter, J. Craig
Assignees
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Publication Number
US-11408020-B2
Publication Date
2022-08-09
Expiration Date
Abstract
The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.
Core Innovation
The invention relates to an in vitro method of joining a set of two or more double-stranded or single-stranded DNA molecules in a single vessel, where adjacent DNA molecules to be joined contain overlapping sequences at their termini. The DNA molecules are contacted in vitro with a mixture including an isolated 5′ to 3′ exonuclease, an isolated DNA polymerase, an isolated ligase, a mixture of dNTPs, and a suitable buffer, and a first assembled dsDNA molecule is formed in a concerted reaction under isothermal conditions at 45–60 °C.
The approach uses overlapping termini that provide predetermined order and orientation through unique distal/proximal sequence identity in the overlap design. It also includes removal of non-homologous terminal sequences so that PCR primer binding regions and rare restriction sites are excluded from the homologous joining outcome. The assembled dsDNA molecule is produced by coordinated activity of the exonuclease, DNA polymerase, ligase, and dNTPs acting in the single-vessel reaction.
The disclosed schemes further include scalability for combinatorial assembly, in which many fragments can be assembled in concert. The document also describes thermocycled and isothermal enzymatic schemes that support a one-step/no-restriction DNA assembly concept, together with kit compositions and compatibility with automation.
Claims Coverage
The independent claim defines an in vitro, single-vessel, isothermal joining method for two or more DNA molecules having overlapping termini, using a specific combination of isolated enzymes and reaction components, and includes formation of a first assembled dsDNA molecule. The inventive features are refined in dependent claims by specifying particular enzyme properties and optional sequence/terminus constraints for the DNA molecules being joined.
Single-vessel joining of overlapping terminal DNA
An in vitro method of joining a set of two or more double-stranded (ds) or single-stranded (ss) DNA molecules, where adjacent DNA molecules to be joined contain overlapping sequences at their termini, by contacting the DNA molecules in a single vessel to form a first assembled dsDNA molecule in a concerted reaction.
Isothermal single-vessel concerted reaction at 45–60 °C
The joining is performed under isothermal conditions at a temperature of 45–60 °C, with amounts effective for joining the two or more DNA molecules in said single vessel.
Isolated 5′ to 3′ exonuclease, isolated DNA polymerase, and isolated ligase with dNTPs
The single-vessel reaction mixture comprises an isolated 5′ to 3′ exonuclease, an isolated DNA polymerase, an isolated ligase, a mixture of dNTPs, and a suitable buffer, to join the overlapping terminal DNA molecules.
Thermostable DNA polymerase and thermostable ligase
The method is performed using a thermostable DNA polymerase and a thermostable ligase.
5′ to 3′ exonuclease lacking 3′ exonuclease activity
The 5′ to 3′ exonuclease does not have 3′ exonuclease activity.
T5 exonuclease as the 5′ to 3′ exonuclease
The method is characterized by using a T5 exonuclease as the exonuclease of (a).
Non-homologous terminus sequences including PCR primer binding regions, vector homology regions, and restriction recognition sites
The method includes joining DNA molecules where some DNA molecules have terminus sequences that are non-homologous to the DNA molecules of interest, including PCR primer binding regions, regions of homology to vector sequences, and restriction enzyme recognition sites.
Across the independent and dependent claims, the core coverage is an in vitro, single-vessel, isothermal joining of DNA molecules with overlapping termini using an isolated 5′ to 3′ exonuclease, an isolated DNA polymerase, an isolated ligase, and dNTPs to form an assembled dsDNA molecule. Dependent claims further narrow or refine the enzyme properties and include specific non-homologous terminus sequences such as PCR primer binding regions, vector homology regions, and restriction enzyme recognition sites.
Stated Advantages
Reproducibility
Speed
Controlled joining
Compatibility with automation
Documented Applications
Combinatorial assembly for optimizing or creating variant nucleic acids for codon usage, control sequences, and protein motif/domain variants
Metabolic pathway optimization
Whole/minimal genome construction
Assembly demonstrations including libraries, with host transformation or in vitro amplification and general screening approaches for libraries
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